Cancer results from the disregulation of signal transduction networks. Thus, quantitative detection, functional assessment and isolation of proteins involved in cancer biology are critically important for molecular analysis of cancer. Short peptide motifs within many of this class of proteins and posttranslational modification thereof play central roles in signal transduction regulation through controlling protein function and interactions. However, there are a very small number of affinity reagents (reagents that bind to a target with high affinity and specificity) to this class of high-value epitopes in cancer analysis. The long-term goal of this project is to develop a powerful technology platform for generating high-performance affinity reagents for short peptide epitopes. The primary products will be novel affinity reagents termed "Epitope Clamps". Our strategy is distinct from many others in that we aim to develop distinct types of affinity reagents, with each type specific to a particular class of short peptide epitopes. This contrasts with the conventional antibody-based approaches where a single, general- purpose platform is used for engineering distinct types of targets. Our innovative protein engineering strategy harnesses the inherent specificity present in the so-called interaction domains and dramatically enhances their affinity and specificity by attaching an "enhancer domain". This makes it possible to generate protein libraries predisposed to binding to a particular motif, and thus it dramatically increases the chance of successfully engineering high- performance affinity reagents. Our proof-of-concept experiments have successfully demonstrated this new paradigm in affinity reagent engineering. These proposed studies will establish the full potential of the Epitope Clamp technology, and will generate high-performance affinity reagents to phosphopeptide epitopes in proteins that are critically involved in cancer biology. The technology and tools developed in this project will make a major impact on molecular analysis of cancer, and more broadly on proteomics and biotechnology. Accurately measuring the amounts of proteins of interest in cells and tissues and assessing their functional state are major technological challenges in molecular analysis of cancer. This application aims to establish a powerful technology platform for facile generation of high- performance reagents that tightly bind to a predefined segment within a protein of interest. We aim to develop such "affinity reagents" to diverse protein segments that carry a chemical signature of activated proteins. This technology will fill a major void in the current technology portfolio for molecular analysis of cancer.